Owing to high incidences of multidrug resistance and challenges posed by complex and long duration treatments, Mycobacterium tuberculosis (Mtb) infections remain a significant clinical burden, which would benefit from development of novel immuno-therapeutic based treatment strategies. Among early immune effectors invariant or innate-like natural killer T (iNKT) cell are attracting attention because of their potential regulatory activity which can shape anti-mycobacterial immune responses. Unlike conventional T cells, iT cells express a semi-invariant T cell receptor and respond rapidly and robustly to molecular patterns presented in the context of MHC class I-like molecules. These iNKT cells, by rapidly producing large amounts of cytokines can either promote or suppress cell-mediated immunity without the need for clonal expansion. However, to date it has been difficult to distinguish in vivo effects due specifically to iNKT cells as opposed to compensatory response of other T cells, and thus the precise roles of iNKT cell in anti-Mtb responses remains unclear. As such reassessing anti-Mtb immune responses with the goal of developing new immunotherapeutic based strategies is needed. This exploratory research proposal addresses this issue by using an alternative, amphibian (Xenopus laevis) tadpole model in combination with Mycobacterium marinum (Mm), a natural pathogen for humans and Xenopus, that in addition is less pathogenic and more amendable to genetic manipulation than Mtb. Importantly, Xenopus tadpoles rely mostly on a few prominent subsets of functionally distinct innate-like (i)T cells, whose development and function are governed by distinct MHC class I-like molecules. This provides a convenient and cost-effective in vivo model system uniquely suited to investigate the roles of iT cells during mycobacterial infections. We recently identified the MHC class I-like gene, XNC4, as the restricting element for a distinct iT cell population (iV?45 T cells). Based on the increased susceptibility of XNC4-deficient tadpoles ablated for the iV?45 T cell subset to Mm infections and other preliminary data, we hypothesize that that iV?45 T cells, critically counteract Mm polarization of macrophages into an anti-inflammatory M2-like through secretion of pro-inflammatory cytokines. Accordingly, we will develop and exploit intravital microscopy and reverse genetic in our in vivo X. laevis tadpole-Mm infectious model to: (1) Investigate how iV?45 T cells affect anti-Mm responses by reverse genetic in the X. laevis tadpole-Mm infection model; (2) Characterize the TCR repertoire and effector profile of iV?45 T cells by trancriptomics; and (3) Define macrophage polarizing functions of iV?45 T cells in vivo by intravital microscopy and reverse genetic. These studies provide us with the unique opportunity to uncover molecular mechanisms governing iT cell activation and regulation which may help to identify novel therapeutic targets. This project will also advance the development of Xenopus as a model for medical immunology and human diseases.